Now that the 50,000 or so genes that make up the human genome have been sequenced, tools are needed to determine when and in what type of tissue those genes are active so as to ascertain their function and role, particularly in disease. This effort, often referred to as “functional genomics” and “proteomics,” is especially important in efforts to discover new drugs since new pharmaceutical agents are being designed to target specific enzymes, receptors, and other proteins. Eventually, proteomic information will be used in clinical diagnostics to help guide treatment selection in the emerging era of “personalized medicine.”
Some believe that the 100,000 human genes may turn out to produce up to a million different protein variants due to post-translational and other modifications. Within the next decade the pharmaceutical industry is expected to identify up to 10,000 proteins against which human therapeutics can be directed. Additional therapeutics, gene modifiers, expression modifiers, and valuable biomolecules also are expected to be developed or identified through the extension of proteomics to the analysis of non-human animals and plants.
Although there may be up to a million different protein variants in humans, only about 10,000-15,000 proteins are expressed in any particular cell type. Thus, for example, liver cells have essentially the same genome as skin cells taken from the same individual, but the two cell populations express substantially different sets of proteins. It is often desirable, therefore, to profile and compare the patterns of proteins (i.e., the “proteome” of a cell) in different cell populations (e.g., diseased and normal tissue; fetal and mature tissue; human and non-human tissue, etc.) to identify targets for drugs.
One common approach to establishing or confirming the association of gene activity with disease is through expression analysis. DNA microarrays are used to survey differential expression patterns of thousands of genes from extracts taken from samples of tissues representing various diseases. If particular genes are expressed in diseased tissue but not in normal tissue they may be relevant as diagnostic markers and targets of pharmaceutical intervention. One disadvantage with this approach is that the sample being tested is disassociated from the tissue from which it was isolated, thereby losing the ability to observe gene expression patterns in the context of the tissue in which the genes are active. Since the morphological relationship is not preserved in microarray analysis, it is hard to know what component of the sample is responsible for the changes observed in gene expression. Also, microarray analysis is usually performed on a homogenized sample of tissue, making it virtually impossible to ascribe expression to a specific cell type, let alone a specific cell.
In situ detection and visualization of proteins traditionally has been accomplished through immuno-histochemistry (IHC). This technique involves the mounting a thin tissue section on the glass slide and visualizing a protein of interest with a detectable antibody that has specific binding affinity for the target protein. Because of certain technical limitations of IHC, only one or two proteins from a single tissue section can be achieved. Also, proteins are still embedded in the tissue and are not presented to the antibodies in the most appropriate way (proteins are not highly denatured) lowering the success rate of the antibody reactivity.
The most widely used method for identifying and measuring proteins and nucleic acids that have been removed from tissue samples is gel electrophoresis. Electrophoresis generally refers to techniques for separating or resolving molecules in a mixture under the influence of an applied electric field. Separation is based on difference in (usually) the size and/or charge of the molecules. Molecules separated by electrophoresis are often visualized by staining with a non-specific dye, such as Coomassie blue (for proteins) or ethidium bromide (for nucleic acids). Such dye staining does not specifically identify individual molecules. Furthermore, ubiquitous dye staining is generally not very sensitive.
More sensitive detection methods exist, such as antibody-based detection for proteins. In particular, immunoblotting, also known as “Western blotting,” is often used to detect gel-separated proteins. This technique uses detectable antibodies specific to the proteins of interest in lieu of a ubiquitous stain. A key imitation of the technique is its low throughput; at most only a handful of proteins can be identified from a single lane of an immunoblot on a single blot, due to overlapping banding patterns and cross reactivity of antibodies with different proteins in the sample. Thus, immunoblotting is typically performed using only one antibody per membrane to ensure specificity.
Though it is possible to strip and re-probe an immunoblot, stripping will also remove protein of the sample that had been bound to the membrane, thus encumbering quantitative analysis of the sample. Moreover, the proportion of each individual protein removed from the membrane by such treatment will vary depending upon the nature of the protein, which further clouds efforts to quantitate the relative amounts of protein initially present in the sample. There remains a clear need to develop blotting techniques that permit larger numbers of antigens to be detected simultaneously from a single test sample.
It would be desirable to have high-throughput approaches for detecting, identifying and comparing large numbers of biomarkers that is relatively inexpensive, can be used by ordinary laboratory personnel, and readily permits the capture, organization, and analysis of the data generated thereby.